The invention relates a soluble immunosuppressive factor produced by cells of a tumor-bearing mammal and to a gene encoding said factor. Screening methods for agents inhibiting or reversing the action of this factor are identified. Immunosuppressive agents and counter-agents are recognized using the invention. The factor may be used to induce immunosuppression. The present invention also relates to the evaluation, selection and treatment of patients with diseases causing progressive immunosuppression. Patients having a disease responsive to immunotherapy, especially cancer patients, are staged or evaluated based on expression levels of proteins affected by immunosuppression. The likelihood of success of such immunotherapy is predicted by the results of staging and evaluation.
Many diseases are characterized by the development of progressive immunosuppression in a patient. The presence of an impaired immune response in patients with malignancies has been particularly well documented. Cancer patients and tumor-bearing mice have been shown to have a variety of altered immune functions such as a decrease in delayed type hypersensitivity, a decrease in lytic function and proliferative response of lymphocytes. S. Broder et al., N. Engl. J. Med., 299:1281 (1978); E. M. Hersh et al., N. Engl. J. Med., 273:1006 (1965); North and Burnauker, (1984).
Many other diseases are also characterized by the development of an impaired immune response. For example, progressive immunosuppression has been observed in patients with acquired immunodeficiency syndrome (AIDS), sepsis, leprosy, cytomegalovirus infections, malaria, and the like. The mechanisms responsible for the down-regulation of the immune response, however, remain to be elucidated.
The immune response is a complex phenomenon. T lymphocytes (T cells) are critical in the development of all cell-mediated immune reactions. Helper T cells control and modulate the development of immune responses. Cytotoxic T cells (killer T cells) are effector cells which play an important role in immune reactions against intracellular parasites and viruses by means of lysing infected target cells. Cytotoxic T cells have also been implicated in protecting the body from developing cancers through an immune surveillance mechanism. T suppressor cells block the induction and/or activity of T helper cells. T cells do not generally recognize free antigen, but recognize it on the surface of other cells. These other cells may be specialized antigen-presenting cells capable of stimulating T cell division or may be virally-infected cells within the body that become a target for cytotoxic T cells.
Cytotoxic or suppressor T cells usually recognize antigen in association with class I Major Histocompatibility Complex (MHC) products which are expressed on all nucleated cells. Helper T cells, and most T cells which proliferate in response to antigen in vitro, recognize antigen in association with class II MHC products. Class II products are expressed mostly on antigen-presenting cells and on some lymphocytes. T cells can be also divided into two major subpopulations on the basis of their cell membrane glycoproteins as defined with monoclonal antibodies. The CD4.sup.+ subset which expresses a 62 kD glycoprotein usually recognizes antigen in the context of class II antigens, whereas the CD8.sup.+ subset expresses a 76 Kd glycoprotein and is restricted to recognizing antigen in the context of Class I MHC.
The CD4.sup.+ subset can be further subdivided into two functionally distinct groups. One group of cells positively influences the immune response of T cells and B cells. The second group of cells induces suppressor/cytotoxic functions in CD8.sup.+ cells.
The definitive T cell marker is the T cell antigen receptor (TCR). TCR-2 is a heterodimer of two disulfide-linked polypeptides (.alpha. and .beta.). TCR-1 is structurally similar to TCR-2, but consists of .gamma. and .delta. polypeptides. Both TCR-1 and TCR-2 are associated with a complex of polypeptides which comprise the CD3 complex.
The TCR found on the surface of all T cells is composed of at least six different subunits which can be divided into three distinct subgroups of proteins. R. D. Klausner et al., Annu. Rev. Cell Biol., 6:403 (1990).
The Ti subunits are responsible for antigen binding, and include the .alpha., .beta., .gamma. and .delta. chains. The heterodimers .alpha..beta. or .gamma..delta. within the receptor complex are responsible for ligand binding. The .alpha..beta. heterodimer is found on most mature T cells and the .gamma..delta. heterodimer is found predominantly on T cells that are located in epithelia.
Another subgroup of proteins which comprise the TCR are the CD3 chains which encompass at least four distinct, but closely related subunits. These subunits are .gamma., .delta., .epsilon. and .zeta.. F. Koning et al., Eur. J. Immunology, 20:299 (1990); R. S. Blumberg et al., Proc. Natl. Acad. Sci. USA, 87:7220 (1990). Diversification of receptor types is the result of segregation of chains of the TCR complex into multiple subunits. Incompletely assembled complexes are degraded, resulting in the surface expression of only completely assembled receptors. R. D. Klausner, New Biol., 1:3 (1989).
In addition to the involvement of the TCR subunit proteins, T cell recognition events lead to signal transduction and appropriate biochemical signals that control cellular responses. The ability of TCR to transduce signals to multiple biochemical cascades is a central event of immune cell activation. The details of this signal transduction pathway, however, are poorly understood. For the TCR, one or more tyrosine (Tyr) kinases likely have an essential role in T cell activation. R. D. Klausner et al., Cell, 64:875 (1991). At least two signal transduction pathways are activated upon stimulation of TCR by an antigen or by monoclonal antibodies directed against either CD3 or the .alpha..beta. heterodimer.
Stimulation of TCR activates a tyrosine kinase. L. E. Samelson et al., Cell, 46:1083 (1986); M. D. Patel et al. J. Biol. Chem., 262:5831 (1987); E. D. Hsi et al., J. Biol. Chem., 264:10836 (1989). Phosphorylation of several proteins with tyrosine residues is induced within seconds of TCR stimulation. C. H. June et al., J. Immunol., 144:1591 (1990). None of the TCR chains possesses intrinsic kinase activity. A member of the Src family of tyrosine kinases designated Fyn, however, coprecipitates with the CD3 complex. L. E. Samelson et al., Proc. Natl. Acad. Sci. USA, 87:4358 (1990). A T cell specific member of the Src family of tyrosine kinases, Lck, is tightly, but non-covalently, associated with the cytoplasmic domain of either a CD4 or CD8 molecule. The extracellular domains of CD4 and CD8 bind to MHC class II and class I molecules, respectively. Upon binding of TCR to an antigen-MHC complex on a presenting cell, the TCR is believed to be brought into close proximity with either a CD4 or CD8 molecule that is capable of independently binding to an appropriate MHC molecule.
TCR also activates a phosphatidylinositol-specific phospholipase C which leads to hydrolysis of phosphatidylinositol-4,5-bis-phosphate. A. Weiss et al., Proc. Natl. Acad. Sci. USA, 81:4169 (1984); J. B. Imboden et al., J. Exp. Med., 161:446 (1985). This leads to the liberation of two second messengers: 1) inositol-1,4,5-tris-phosphate which is responsible for transient Ca.sup.2+ mobilization; and 2) diacylglycerol which is a potent activator of protein kinase C. B. Berridge et al., Nature, 341:197 (1989).
The cytoplasmic domain of the TCR .zeta. chain is sufficient to couple stimulation of the receptor with the signal transduction pathways. B. A. Irving et al., Cell, 64:891 (1991). When a chimeric protein linking the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of the .zeta. chain was constructed, the chimeric protein activated the appropriate signal transduction pathways in the absence of CD3.gamma., .delta., and .epsilon.. Therefore the role of .zeta. is apparently to couple the TCR to intracellular signal transduction mechanisms.
Another set of proteins that are related to signal transduction are the NF-.kappa.B/rel transcription factors. The NF-.kappa.B transcription activator is a multiprotein complex. The NF-.kappa.B transcription activator appears to have specialized in the organism to rapidly induce the synthesis of defense and signalling proteins upon exposure of cells to a wide variety of agents including cytokines, double-stranded RNA, T cell mitogens, DNA damaging agents, protein synthesis inhibitors, parasites, viruses and viral transactivators. A common denominator of the agents that activate NF-.kappa.B is that they either signal or represent a threat to cells and the organisms.
NF-.kappa.B is particularly suited to rapidly activate gene expression because (1) it does not require new protein synthesis, (ii) a simple dissociation reaction triggers activation, (iii) NF-.kappa.B actively participates in cytoplasmic-nuclear signalling and (iv) it is a potent transactivator.
NF-.kappa.B is involved in the inducible expression of the T cell growth factor interleukin-2 and a component of its high affinity receptor suggesting that NF-.kappa.B can be a growth regulator. There is indeed a good correlation between the proliferative state of T cells and the state of NF-.kappa.B activity.
Three protein subunits, I.sub.k B, p50 and p65 control the biological functions of NF-.kappa.B. I.sub.k B is a 35-43 kDa subunit which inhibits the DNA-binding of NF-.kappa.B and serves to retain NF-.kappa.B in an inducible form in the cytoplasm of unstimulated cells. Upon stimulation of cells, I.sub.k B dissociates from the inactive complex with p65 and p50. The released p50-p65 complex heterodimer then migrates into the nucleus and trans-activates genes. Constitutive expression of the IL-2 receptor .alpha. gene in hybrids between a T-cell and myeloma cell line depends solely on the presence of the heterodimer. Only p65 appears to bind I.sub.k B. Within cells, I.sub.k B could be released by modification of either I.sub.k B, p65, or both.
Rel proteins are believed to be transcriptional activators, are capable of forming heterodimers with p50, and are sequence-specific DNA binding proteins which are capable of recognizing kB motifs. I.sub.k B is a member of a family of proteins that share homologies and apparently play a similar role in the cell. Both p50 and p65 have homologies to the rel and dorsal proteins. The I.sub.k B -family and rel-family therefore comprise related proteins which are known to be involved in cytoplasmic/nuclear signalling.
Other information on the NF-.kappa.B transcription activator and its relationship to the rel proteins may be found in Baeuerle, P. A. (1991), "The inducible transcription activator NF-.kappa.B regulation by distinct protein subunits", Biophysica Acta 1072:63-80.
Chemotherapy as a treatment modality has not been successful for all types of cancer. The identification and isolation of soluble mediators of the immune response has heightened interest in the development of clinical trials using immunotherapy as an alternative form of treatment. For example, biological response mediators (BRM) such as interleukin-2 (IL-2), a lymphokine produced by helper T cells, stimulates the growth of T cells that bear IL-2 receptors, either in vivo or in vitro. It also activates (enhances) the anti-tumor function of natural killers (NK) cells (Lotze et al. 1981). NK cells also express the .zeta. chain. The in vitro incubation of resting lymphocytes in supernatants containing IL-2 for three to four days results in the generation of lymphocytes capable of mediating the lysis of fresh tumor cells, but not of normal cells. These lysing cells are referred to as lymphokine activated killer (LAK) cells. I. Yron et al., J. Immunol., 125:238 (1980); M. T. Lotze et al., Cancer Res., 41:4420 (1981); and S. A. Rosenberg et al., J. Natl. Cancer Inst., 75:595 (1985).
A method for the activation of T lymphocytes to generate T-activated killer cells (T-AK) has been described as taking lymphocytes by leukophoresis or from peripheral blood, and stimulating said cells with a monoclonal antibody (MAb) to a T cell surface receptor such as anti-CD3 (soluble or solid phase bound). The T cells can be stimulated with or without the addition of one or more cytokines such as IL-2. Alternatively, T cells can be purified before stimulation with the MAb to a surface receptor. Experimentation with T-AK cells has demonstrated that CD8.sup.+ cells are responsible for the non-MHC restricted cytolytic activity seen in these cultures. P. M. Anderson et al., J. Immunol., 142:1383 (1989); C. M. Loeffler et al., Cancer Res., 51:2127 (1991). The ability of IL-2 to expand T lymphocytes having immune reactivity and the ability to lyse fresh autologous, syngeneic, or allogeneic natural killer (NK) cell-resistant tumor cells, but not normal cells, has resulted in the development of cell transfer therapies, such as autologous adoptive immunotherapy.
Typical adoptive immunotherapy involves the administration of immunologically active cells to an individual for the purpose of providing a beneficial immunological effect such as reduction or control of cancer. The immunologically active cells are typically taken by venipuncture or leukophoresis either from the individual to be treated, termed autologous treatment, or from another individual, termed an allogeneic treatment. The lymphocytes are then cultured to increase their number and to activate their antitumor activity, and then infused back into the patient. Thus, the majority of conventional efforts in adoptive immunotherapy are directed at expanding cell numbers in vitro followed by infusion back into the patient.
Animal experiments involving the transfer of immunologically active cells from healthy animals to animals with cancerous tumors have indicated that adoptive immunotherapy can elicit an antitumor effect in certain tumor models with a high degree of effectiveness. The administration of IL-2 together with LAK cells has proven effective in the treatment of a variety of murine malignancies. The transferred LAK cells also proliferate in vivo as a result of IL-2 treatment. Human clinical trials have demonstrated that LAK cells plus IL-2 or IL-2 alone can be effective in mediating the regression of established metastatic cancer in selected patients. S. A. Rosenberg, "Immunotherapy of Patients with Advanced Cancer Using Interleukin-2 Alone or in Combination With Lymphokine Activated Killer Cells," in Important Advances in Oncology 1988: J. B. Lippincott Co., 217, (1988).
However, the success of adoptive immunotherapy has been limited by the large number of cells required in the therapy, the large amount of culture medium and large number of hours involved in culturing cells to develop LAK activity, the length of time sufficient LAK activity must be maintained for the desired therapeutic efficacy, the time involved in clinical treatment and the side effects of treatment. Improvements in the in vitro culturing process have been sought in order to increase the efficacy of adoptive immunotherapy. Cells cultured in IL-2 and/or monoclonal antibodies against the antigen receptor complex CD3 (anti-CD3 MAb) have been found to induce proliferation of a greater number of T cells, which demonstrate an increased anti-tumor activity. P. M. Anderson et al. Cancer Immunol. Immunother., 27:82 (1988); P. M. Anderson et al., J. Immunol., 142:1383 (1989); and A. C. Ochoa et al., Cancer Res., 49:963 (1989).
Adoptive immunotherapy of renal adenocarcinoma in the murine RENCA model using IL-2-activated killer cells in conjunction with chemotherapy showed some success in reducing tumors, but the effect was suspected to be cytokine mediated. Wiltrout, Progress in Neuro Endocrin Immunology, 4:154 (1991).
There has been limited success with efforts to activate in vivo antitumor mechanisms. Only a minority of patients receiving high doses of IL-2 experienced therapeutic effects, and significant toxicity is observed. The direct infusion of anti-CD3 monoclonal antibody alone inhibits nonspecific antitumor function in mice. D. W. Hoskin et al., Cancer Immunol Immunother., 29:226 (1989). Based on the positive results in murine models, direct infusion of anti-CD3 has been attempted in humans. Although patients who have directly received the anti-CD3 MAb designated OKT3 have experienced the activation of some T cells in vivo, the toxicity of intravenous OKT3 reaches the maximum tolerated dose (MTD) at low doses before it starts showing some immune efficacy. W. Urba et al., Cancer Res., Cancer Res. March 1991. It is believed that the free OKT3 is responsible for the majority of these toxic effects.
Due to inadequacies of treatment with single modalities, synergism has been sought. Flavone acetic acid (FAA) is a cytokine-inducing synthetic drug which showed a synergistic therapeutic effect with IL-2 in reducing RENCA murine tumors. Antitumor effects of flavonoids and cytokines in combination with IL-2 are therefore promising. FAA is a biological response modifier (BRM) which has been shown to augment systemic natural killer (NK) cell activity and to synergize with IL-2 for the successful treatment of advanced RENCA. Wiltrout et al., J. Immunol., 140:3261 (1988); U.S. Pat. No. 5,096,707 and 5,061,488. Cytokines (TNF, .alpha.-IFN, and .gamma.-IFN) induced by FAA have been implicated in the therapeutic synergy with rIL-2. CD8.sup.+ T-lymphocytes are critical in mediating this synergistic effect. FAA induces cytokine genes in vitro. In this model, immunosuppressive T-cells are induced during the progression of the RENCA tumor. Gregarian et al., Cancer Immunol Immunother 31:325, 31:335 (1990).
T lymphocytes from hosts bearing tumors exhibit decreased immune function in a variety of in vitro tests. R. Lafreniere et al., J. Surg. Oncol., 43:8 (1990); R. J. North et al., J. Exp. Med., 159:1295 (1984); M. Sarzotti et al, Int. J. Cancer, 39:118 (1987). It has been observed that before the decrease in the immune responsiveness in peripheral blood lymphocytes, T lymphocytes infiltrating a tumor exhibit poor cytotoxic activity against autologous or allogeneic tumor cells. E. F. Klein et al., 1980, In: Contemporary Topics in Immunobiology, I. P. Witz and M. G. Hanna, Jr., eds. Plenum Press, N.Y., p 79-107; B. M. Vose et al., J. Cancer, 44:846 (1981).
The molecular basis of the decreased immune responsiveness of the T cells derived from tumor-bearing hosts is poorly understood. It has been proposed that decreased immune responsiveness of the T cells is caused by the development of suppressor lymphocytes. S. B. Mizel et al., Proc. Natl. Acad. Sci. USA, 77:2205 (1980); C. C. Ting et al., Int. J. Cancer, 24:644 (1979). Another proposal is that responsive T cell clones are deleted. S. Webb et al., Cell, 63:1249 (1990). It has also been proposed that decreased immune responsiveness of the T cells is the result of the induction of T cell anergy. M. K. Jenkins et al., J. Exp. Med., 165:302 (1987). Others have suggested that the major alteration in the immune response is produced by a modification in the presentation of the antigen which results in an inadequate response of the CD4.sup.+ helper T lymphocytes. These data have been strengthened by the observation that tumor cells transfected with cytokine genes induce a protective antitumor response, and result in an immunological memory response. E. R. Fearon et al., Cell, 60:397 (1990).
In an in vivo tumor model, the progressive growth (&gt;26 days) of a subcutaneous implant of murine colon carcinoma designated MCA-38 resulted in decreased lytic function by the CD8.sup.+ T lymphocytes, a decrease which was associated with decreased expression of mRNA for tumor necrosis factor .alpha. (TNF-.alpha.) and granzyme B, and the complete loss of the ability of adoptively transferred cells to mediate an antitumor effect in vivo (Loeffler et al. 1992, J. Immunol.149:949). However, proliferation, lymphokine production, and lymphokine receptor upregulation in CD4.sup.+ T cells were comparable in normal and tumor-bearing mice. Cells with suppressor function were not detected, nor was the production of transforming growth factor-.beta. (TGF-B) detected in the lymphocytes from tumor-bearing mice or the MCA-38 tumor cells.
Augmentation of the immune response in immune compromised patients via infusions of lymphokines and/or adoptive immunotherapy has met with variable and limited success. Methods are needed to improve this type of treatment. A need exists for effective methods of measuring the progression of immunosuppression so that attempts at augmenting the immune system in an immunosuppressed patient can be effectively timed. A need also exists for a method by which a patient's level of immunosuppression is estimated and used to accurately predict the likelihood of a patient's response to therapy. The patient's therapy can then be developed in a systematic fashion. A method is needed by which a clinician can determine whether a patient's T lymphocytes will be capable of activation and, thus, whether autologous adoptive immunotherapy will likely be efficacious.
A need exists for a method by which the immunosuppressed state of T lymphocytes during disease progression can be circumvented or reversed so that the T cell immune response in the patient can develop or be augmented. A need also continues to exist for a method of screening for immunosuppressive agents and agents that reverse or inhibit immunosuppression.
There is a need to detect the presence of tumors, in particular early in the development of a tumor, so that treatment effectiveness is enhanced. Also, improved methods for staging of the disease would facilitate choice of the most appropriate treatment modalities. There is also a need to test the effectiveness of treatment modalities prior to clinical trials, and as adjuncts to clinical trials.
The present invention addresses limitations in the art for the detection, monitoring, and reversal of immunosuppression, and of diseases generally characterized by immunosuppression, for example, cancer.